X-RAY STRUCTURE OF CYCLODEXTRIN GLYCOSYLTRANSFERASE COMPLEXED WITH ACARBOSE - IMPLICATIONS FOR THE CATALYTIC MECHANISM OF GLYCOSIDASES

Crystals of cyclodextrin glycosyltransferase (CGTase) from Bacillus ci rculans strain 251 were soaked in buffer solutions containing the pseu dotetrasaccharide acarbose, a strong amylase- and CGTase inhibitor. Th e X-ray structure of the complex was elucidated at 2.5-Angstrom resolu tion with a final crystallographic R value of 15.8% for all data betwe en 8.0 and 2.5 Angstrom. Acarbose is bound near the catalytic residues Asp229, Glu257, and Asp328. The carboxylic group of Glu257 is at hydr ogen bonding distance from the glycosidic oxygen in the scissile bond between the B and C sugars (residue A is at the nonreducing end of the inhibitor). Asp328 makes hydrogen bonds with the 4-amino-4,6-dideoxyg lucose (residue B), and Asp229 is in a close van der Waals contact wit h the C1 atom of this sugar. From this we conclude that in CGTase Glu2 57 acts as the proton donor and Asp229 serves as the general base or n ucleophile, while Asp328 is involved in substrate binding and may be i mportant for elevating the pK(a) of Glu257. On the basis of these resu lts it appears that the absence of the C6-hydroxyl group in the B suga r is responsible for the inhibitory properties of acarbose on CGTase. This suggests that the C6-hydroxyl group of this sugar plays an essent ial role in the catalytic mechanism of CGTase. The binding mode of aca rbose in CGTase differs from that observed in the complex of pancreati c alpha-amylase with acarbose where the catalytic Glu was found to be hydrogen bonded to the glycosidic nitrogen linking the A and B residue s [Qian, M., Haser, R., Buisson, G., Duee, E., and Payan, F. (1994) Bi ochemistry 33, 6284-6294].